The adipokinetic hormones of Odonata: A phylogenetic approach

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Journal of Insect Physiology 51 (2005) 333–341 www.elsevier.com/locate/jinsphys

The adipokinetic hormones of Odonata: A phylogenetic approach Gerd Ga¨de, Heather G. Marco Zoology Department, University of Cape Town, Private Bag, Rondebosch 7701, South Africa Received 8 November 2004; accepted 30 December 2004

Abstract Adipokinetic neuropeptides from the corpora cardiaca of the major families of all three suborders of the Odonata were identified by one or more of the following methods: (1) Isolation of the peptides from a methanolic extract of the corpora cardiaca by liquid chromatography, peak monitoring by fluorescence of the Trp residue and comparison of the retention time with those of known synthetic peptides of Odonata. (2) Hyperlipaemic bioassays of the HPLC-generated fractions either in Locusta migratoria or, in a few cases, in Anax imperator or Orthetrum julia. (3) Sequencing of the isolated, bioactive HPLC fraction by Edman degradation. (4) Mass spectrometric measurement of the isolated, bioactive fraction. Sequence assignment revealed that the investigated Odonata species always contain only one adipokinetic peptide. This is always an octapeptide. The suborder Zygoptera contains the peptide code-named Psein-AKH, the Anisozygoptera and the families Aeshnidae, Cordulegastridae and Macromiidae of the Anisoptera contain Anaim-AKH, whereas Gomphidae, Corduliidae (with the exception of Syncordulia gracilis) and Libellulidae contain Libau-AKH; one species of Libellulidae has Erysi-AKH, a very conservative modification of Libau-AKH (one point mutation). When these structural data are interpreted in conjunction with existing phylogenies of Odonata, they support the following: (1) Zygoptera are monophyletic and not paraphyletic. (2) Anisozygoptera and Anisoptera are sister groups and contain the ancestral Anaim-AKH which is independently and convergently mutated to Libau-AKH in Gomphidae and Libellulidae. (3) The Corduliidae are of special interest. Only Corduliidae sensu stricto appear to contain Libau-AKH, other species placed into this family by most authorities contain the ancestral Anaim-AKH. Possibly, assignments of AKHs can untangle the paraphyly of this family.

r 2005 Elsevier Ltd. All rights reserved. Keywords: Adipokinetic neuropeptides; Liquid chromatography; Mass spectrometry; Hyperlipaemic bioassay; Phylogeny of Odonata

1. Introduction The vertebrate hypothalamo-hypophysial system is well-known to endocrinologists. Analogous structures Corresponding author. Tel.: +27 21 650 3615; fax: +27 21 650 3301. E-mail addresses: [email protected] (G. Ga¨de), [email protected] (H.G. Marco).

0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.12.011

are found in some invertebrate groups: the corpora cardiaca in the heads of insects and the X-organ–sinus gland complex in the eyestalks of crustaceans are neuroendocrine organs that have interested scientists for many decades. The corpora cardiaca and the X-organ synthesise hormones that structurally belong to the same peptide family, viz. the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family of peptides, so named after the first

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G. Ga¨de, H.G. Marco / Journal of Insect Physiology 51 (2005) 333–341

fully characterised members and their most prominent functions (see Ga¨de, 1996). RPCH causes pigment aggregation in epidermal cells of crustaceans, resulting, thus, in blanching of the integument; the insect AKHs, on the other hand, regulate the level of circulating metabolites (lipids, carbohydrates, proline) by activating phosphorylases or lipases in fat body cells (see Ga¨de, 1997, 2004; Ga¨de and Auerswald, 2003). Whereas the octapeptide code-named Panbo-RPCH is the only member of this peptide family found in all crustaceans examined thusfar (see Rao, 2001; Ga¨de and Marco, 2005), more than 35 isoforms, including Panbo-RPCH, have been identified by primary sequence from various insect species (Ga¨de, 1997; Ga¨de et al., 1997a, 2003a). The AKH/RPCH peptides are characteristically blocked at the N-terminus by pGlu and at the C-terminus by carboxyamide, have a chain length of 8–10 amino acids with at least two aromatic residues at positions 4 (mostly Phe) and 8 (always Trp), and a Gly residue at position 9 (see Ga¨de, 1996; Ga¨de et al., 1997a). Analysis of the primary structures of the AKH peptides has established a certain order- or family specificity, and such data have been used as additional information to aid in the construction of phylogenetic trees (Ga¨de, 1989; Ga¨de et al., 1994a, 1997b, 2003b). The phylogeny of the order Odonata is still under dispute. Extant Odonata are subdivided into the three suborders Zygoptera (damselflies), Anisozygoptera (represented by only one species in Japan, Epiophlebia superstes, and one in the Himalayas, E. laidlani), and Anisoptera (dragonflies). Most phylogenetic relationships of Odonata are based on morphological characters, often even exclusively on wing venation (see Trueman, 1996). For a long time it was accepted that the ancestral odonates were members of the Zygoptera (see Fraser, 1957). More recently, however, it was questioned whether the extant Zygoptera are a monophyletic taxon or are rather paraphyletic (see Rehn, 2003). Bechly (1996), studying mainly wing venation, suggested that Zygoptera are monophyletic and places the ancestral odonate taxon as anisozygopteroids. Monophyly of the Zygoptera is supported by other morphological evidence in the investigations of

Fleck et al. (2001) and Rehn (2003). The Anisoptera, which are viewed by most authors as a monophyletic entity, are then the sister taxon of the extant Anisozygoptera. However, the relationships between the various families of Anisoptera (about 6–15 families exist according to the different authorities) is not resolved (see, for example, Trueman, 1996; Bechly, 1996; Misof et al., 2001). Phylogenetic studies on Odonata using molecular markers (mitochondrial genes) are still scarce. One set of studies analyses only certain genera of Zygoptera (Ischnura, Chippendale et al., 1999; Calopteryx, Misof et al., 2000) or of Anisoptera (Libellula, sensu lato, Kambhampati and Charleton, 1999). Another set of data uses mitochondrial 12S and 16S rRNA gene fragments to unravel the phylogeny of the Anisoptera (Misof et al., 2001). The latter authors cautiously confirm the recent phylogenies which are based on morphological characters (Bechly, 1996) with their molecular data, but are also unable, as yet, to achieve good phylogenetic signals at the interfamily level, although they are optimistic that by adding more taxa these ambiguities would be resolved. In the light of these data, it may be useful to include a totally different data set to the present morphological and molecular ones and to explore whether such an exercise is useful to obtain more clarity about the odonate phylogeny. For this purpose, we chose to analyse the AKH peptides. The following is known with respect to AKHs in Odonata: in 1990 it was shown that the corpora cardiaca of the libellulid Anisoptera, Libellula auripennis, contain and synthesise a blocked, uncharged octapeptide which was clearly a member of the AKH/RPCH family of peptides and had a pronounced hyperlipaemic effect when injected into the dragonfly (Ga¨de, 1990; for primary structure see Libau-AKH in Table 1). An octapeptide with a slightly different structure was found in the aeshnid Anisoptera, Anax imperator (Ga¨de et al., 1994b; Anaim-AKH in Table 1); in the two coenagrionid Zygoptera, Pseudagrion inconspicuum and Ischnura senegalensis yet a different octapepide was identified (Janssens et al., 1994; Psein-AKH in Table 1). Both peptides cause lipid mobilisation in their respective

Table 1 Adipokinetic hormones of Odonata: their primary sequences (with amino acid substitutions indicated in bold) and mass data (protonated and cationised masses) Peptide code-namea

Psein-AKH (3) Anaim-AKH (1) Libau-AKH (2) Erysi-AKH (4) a

Primary structure

pEVNFTPGWamide pEVNFSPSWamide pEVNFTPSWamide pELNFTPSWamide

Mass data [M+H]+

[M+Na]+

[M+K]+

930 946 960 974

952 968 982 996

968 984 998 1012

The number in brackets corresponds to the numbered HPLC peaks in Fig. 1E.

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species. The fourth octapeptide identified from Odonata is a very conservative modification of the peptide Libau-AKH and occurs in the libellulid Anisoptera, Erythemis simplicicollis where it increases the lipid concentration in the haemolymph (Ga¨de and Kellner, 1999; see Erysi-AKH in Table 1). By using a number of biochemical and biological methods, the present study isolates and determines the AKH peptides from species of the major families of extant odonate orders collected in Africa, Europe, United States of America and Japan. The structural data are then used to analyse existing data on phylogenies and interpret how far the AKH data support or refute phylogenetic trends.

2. Materials and methods 2.1. Insects For heterologous bioassays (see below) adult male locusts, Locusta migratoria, were used 14–25 days after adult emergence; they were reared as outlined previously (Ga¨de, 1991). Adult specimens of various species of Odonata of unknown age were caught by netting in the field, kept on ice in paper envelopes to prevent flapping of the wings and to keep the metabolic rate low. In two cases (Anisoptera: Aeshna cyanea and Somatochlora metallica) last instar larvae were collected. The species investigated in this study are listed below. In the Western Cape Province of South Africa we collected: Chlorolestes tesselatus, Aeshna miniscula, Anax speratus and Syncordulia gracilis at the Palmiet River near Grabouw, Chlorolestes umbratus, Platycypha fitzsimonsi, Ellatoneura frenulata and Crocothemis sanguinolenta in the Kogelberg Nature Reserve, Crocothemis erythraea, Orthetrum julia and Trithemis arteriosa around Cape Town. In the KwaZulu-Natal Province of S. Africa: Allocnemis leucosticta, Ceratogomphus pictus, Orthetrum julia, Trithemis dorsalis, T. stictica and Lestes tridens around Pietermaritzburg; Chlorolestes fasciatus, Lestes plagiatus, Aeshna subpupillata, A. miniscula and Anax speratus in Giants Castle Nature Reserve; Phyllomacromia bifasciata at Charters Creek. In the Northern Cape Province of S. Africa: Ictinogomphus ferox in the Kalahari Gemsbok National Park (Now: Kgalagadi Transfrontier Park). In Mpumalanga Province: Brachythemis leucosticta and Pantala flavescens at Skukuza Camp of the Kruger National Park and Phaon iridipennis and Platycypha ( ¼ Chlorocypha) caligata at the Sabie River in Kruger National Park. Prof. M. Samways and P. Grant (University of Stellenbosch) helped with the identification.

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On the island of Mauritius, specimens of the genera Trithemis (very likely T. annulata) and Tramea (very likely T. basilaris) were collected. In Europe Calopteryx splendens, Aeshna cyanea, A. juncea, A. mixta and Orthetrum cancellatum were collected near Bayreuth, Germany in collaboration with Dr. W. Vo¨lkl (University of Bayreuth), larvae of A. cyanea and Somatochlora metallica were collected near Bonn, Germany and identified by Dr. B. Misof (Alexander Ko¨nig Museum, Bonn); C. splendens and Aeshna grandis were collected in the vicinity of Kilkenny, Ireland. In Japan Anotogaster sieboldi and Sympetrum infuscatum were collected near Tsukuba, specimens of Epiophlebia superstes were collected by Dr. K. Inoue near Kyoto, the corpora cardiaca were dissected by Dr. T. Okuda (National Institute of Agribiological Sciences, Tsukuba) and the dried extract was sent to us. In USA Sympetrum vicinum was collected around Amherst, MA by Prof. J. Kunkel (University of Massachusetts, Amherst) who also dissected the glands and sent them to us. Specimens of Libellula vibrans, Pachydiplax longipennis, Anax junius, Nasiaeshna pentacantha and Aphylla angustifolia were caught near Lafayette, LA; the help of Dr. J.H. Spring is gratefully acknowledged. 2.2. Isolation of peptides from corpora cardiaca and structural analyses After transportation of the specimens to the laboratory, corpora cardiaca were dissected as fast as possible into 80% methanol, and the material was stored at 20 1C. Methanolic extracts were prepared as described previously (Ga¨de et al., 1984). The dried material was either taken up in water for bioassays (see below), or it was dissolved in 15% acetonitrile containing 0.1% trifluoroacetic acid (TFA) for reversed-phase high performance liquid chromatography (RP-HPLC) using equipment and methods described previously (Ga¨de, 1985). In brief, CC extracts were separated on a Nucleosil 100 C18 column (4.6 mm  25 cm) with a linear gradient of 43–53% solvent B in 20 min at a flow rate of 1 ml/min (Solvents: A ¼ 0:11% TFA in water; B ¼ 0:1% TFA and 60% acetonitrile in water; see also legend to Fig. 1). AKH fractions were identified as those having lipid-mobilising activity when aliquots were injected into locusts (or dragonflies), or, those fractions that displayed a distinct fluorescence peak (at 276 nm for excitation and 350 nm for emission to monitor the characteristic Trp residue of peptides from the AKH family). Such fractions were, in the majority of cases, analysed further as follows to obtain structural information: (i) the material was digested with the enzyme pyroglutamate aminopeptidase as outlined previously (Ga¨de et al., 1988) and the resulting mixture of digested and undigested peptides was again separated by

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Fig. 1. Isolation of the AKH in Odonata by reversed-phase high performance liquid chromatography (RP-HPLC). Separation was attained with a Nucleosil 100 C18 column (4.6 mm  25 cm) and a linear gradient of 43–53% solvent B in 20 min at a flow rate of 1 ml/min (Solvents: A ¼ 0:11% TFA in water; B ¼ 0:1% TFA and 60% acetonitrile in water). Eluting AKHs were detected via fluorescence at 276 nm excitation and 350 nm emission. Elution profiles of methanolic extracts of corpora cardiaca from (A) 1 adult Aeshna cyanea, (B) 6 A. cyanea larvae, (C) 1 adult Crocothemis sanguinolenta and (D) 11 adult Platycypha fitzsimonsi. (E) Retention times of synthetic compounds corresponding to AKHs that occur in Odonata species. Peak numbers 1–4 relate to Anaim-AKH, Libau-AKH, Psein-AKH and Erysi-AKH, respectively. The histogram in (A) shows the increase in circulating lipid concentrations in locusts (n ¼ 4; mean7SD) after injection of the equivalent of 0.2 gland’s worth of material from 1 min HPLC fractions; the injection of water did not cause a significant increase in the lipid concentration (data not shown).

RP-HPLC as above. The deblocked peptide was subjected to automated Edman degradation with equipment described previously (Ga¨de and Kellner, 1999). (ii) Aliquots of the peak material was analysed by mass spectrometry using a Voyager Elite matrixassisted laser desorption/ionisation (MALDI) instrument (Perseptive Biosystems, Inc., Framingham, MA, USA). Samples were prepared in a-cyano-4-hydroxycinnamic acid, and spectra in the range of 600–2000 Da were acquired in positive, linear mode.

( ¼ total lipids) in the haemolymph, were performed as described earlier (Ga¨de, 1980). In a few instances, adult dragonflies (either A. imperator or Orthetrum julia) were used as bioassay insects in place of locusts and the lipid bioassay was performed as outlined previously (Ga¨de et al., 1994b).

3. Results 3.1. Identification of AKH peptides

2.3. Bioassays The heterologous bioassays with locusts, in which one measures the increase of vanillin-positive material

To illustrate which methods were employed to identify the AKH peptides from the corpora cardiaca of various odonate species, Fig. 1 shows as an example

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the fluorescence elution profile from three representative species, Aeshna cyanea (adult and larval glands; Fig. 1A and B), Crocothemis sanguinolenta (Fig. 1C) and Platycypha fitzsimonsi (Fig. 1D). In addition, the elution profile is shown for the four known AKH peptides of Odonata, using synthetic compounds (Fig. 1E). For A. cyanea, 1 min fractions were collected and an aliquot of each fraction, representing 0.2 gland equivalents, was injected into locusts to test for an adipokinetic response. As depicted in the histogram in Fig. 1A, only the fractions with a retention time around 11.2 min were active. At 11.2 min a distinct fluorescence peak occurred and the synthetic peptide Anaim-AKH run under identical conditions had the same retention time (Fig. 1E). When an aliquot of the peak material of A. cyanea was subjected to MALDI mass spectrometry, clear mass peaks at m/z 968 and 984 Da were detected representing the sodium [M+Na]+ and potassium [M+K]+ cationised molecules (data not shown, but see Table 1); thus the mass of the peptide is 945 Da. When the active peak material from 5 glands of A. cyanea was deblocked after treatment with pyroglutamate aminopeptidase and separated by HPLC, the deblocked peptide yielded the following primary sequence by Edman degradation studies (pmol in brackets): Val (15.6)–Asn (10.0)–Phe(10.3)–Ser (7.8)–Pro(6.2)–Ser(5.0)–Trp(2.0). Thus, the peptide had the same primary structure as Anaim-AKH (see Table 1). The larval corpora cardiaca of A. cyanea produced a fluorescence peak at the same retention time as the extract from adult corpora cardiaca (compare Figs. 1B with A). The gland material from C. sanguinolenta, which was very little and came from one corpus cardiacum, and from P. fitzsimonsi produced fluorescence peaks with retention times identical to synthetic Libau-AKH and PseinAKH, respectively (compare Figs. 1C and D with E). The mass data of the two peaks confirmed the interpretation that they were identical to Libau-AKH and Psein-AKH, respectively (data not shown; for masses see Table 1). For more than 50% of the species analysed, it was possible to collect sufficient gland material and to subject the material to the full spectrum of diagnostic methods as outlined above. For other species where less material was available, chromatographic behaviour on HPLC in conjunction with mass spectrometric results was used for identification. In about 30% of the species (indicated in Table 2) only one to three glands were available for the study; here the peptide was solely identified by retention time and co-elution with the synthetic odonate peptides (see Fig. 1E). Since the four peptides known to occur in Odonata are easily distinguished by their retention times on HPLC (see Fig. 1E), we feel confident that our interpretation of the peptide sequence is correct.

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3.2. Sequences of Odonata AKH peptides Table 2 summarises the results for all investigated species. It is evident that only one AKH peptide, an octapeptide, is found in each species. Moreover, all species of the suborder Zygoptera contain the peptide Psein-AKH. The representative species of the suborder Anisozygoptera, E. superstes, as well as members of the families Aeshnidae, Cordulegastridae and Macromiidae of the Anisoptera have the peptide Anaim-AKH. The families Gomphidae and Libellulidae of the Anisoptera contain the peptide Libau-AKH; with the one exception of Erysi-AKH in the libellulid Erythemis simplicicollis as reported previously (Ga¨de and Kellner, 1999). A somewhat confusing pattern emerged in the anisopteran family Corduliidae: S. gracilis has Anaim-AKH but the larval glands of S. metallica contain Libau-AKH.

4. Discussion 4.1. AKH peptide distribution in Odonata With about 5000 described species worldwide, the Odonata are a medium species-rich order; these species are more or less equally distributed between the main suborders, Zygoptera and Anisoptera. The current data on primary structures of adipokinetic neuropeptides, however, show an unequal distribution between the two suborders: only one AKH form is found for the Zygoptera, and three are found for the Anisoptera. In general, AKH peptides show a low diversity in Odonata. Despite having analysed mostly two species from the major families of Zygoptera, only one octapeptide, i.e. Psein-AKH, is present in this suborder. Similarly, when we analysed species (at least three) of the three most species-rich families of Anisoptera, i.e. the Aeshnidae, Gomphidae and Libellulidae which together comprise about 75% of the extant anisopteran species, our results indicated, again, low diversity in AKH peptide structures. Each species contains an octapeptide, but three different ones are found, viz. Anaim-AKH, Libau-AKH and Erysi-AKH. In the entire order of Odonata, no decapeptide AKH has been found. This pattern of AKH peptide distribution is quite different to that encountered in the Caelifera, where one taxon may have up to three different AKH peptides and these vary from octa- to decapeptides (see, for example, Siegert et al., 2000). Although not systematically studied yet with respect to AKH peptides, Diptera also contain octa- and decapeptides in one taxon (Jaffe et al., 1989), and Hymenoptera also have octa- and decapeptides, although not in one species (Lorenz et al., 2001). The situation in Odonata is more reminiscent of what is encountered in the Ensifera, where only two different octapeptides occur in the entire clade (Ga¨de et al., 2003b).

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Table 2 Species of Odonata investigated in this study and the code-name of the adipokinetic peptide assigned Sub-order

Family

Species

Peptide

Zygoptera

Platycnemididae Protoneuridae Coenagrionidae

Allocnemis leucosticta Ellatoneura frenulata Pseudagrion inconspicuumb Ischnura senegalensisb Lestes tridens L. plagiatus Chlorolestes fasciatus C. tessellatus C. umbratus Platycypha caligata Platycypha fitzsimonsi Calopteryx splendens Phaon iridipennis

Psein-AKHa Psein-AKH Psein-AKH Psein-AKH Psein-AKHa Psein-AKHa Psein-AKH Psein-AKHa Psein-AKH Psein-AKHa Psein-AKH Psein-AKH Psein-AKH

Lestidae Synlestidae

Chlorocyphidae Calopterygidae Anisozygoptera

Epiophlebiidae

Epiophlebia superstes

Anaim-AKHa

Anisoptera

Aeshnidae

Aeshna cyaneac A. grandis A. juncea A. mixta A. minuscule A. subpupillata Nasiaeshna pentacantha Anax imperatorb A. speratus A. junius Ceratogomphus pictus Ictinogomphus ferox Aphylla angustifolia Anotogaster sieboldi Phyllomacromia bifasciata Syncordulia gracilis Somatochlora metallicad Brachythemis leucosticta Crocothemis erythraea C. sanguinolenta Orthetrum julia O. cancellatum Pachydiplax longipennis Pantala flavescens Sympetrum infuscatum S. vicinum Trithemis arteriosa T. dorsalis T. stictica T. annulata Tramea basilaris Libellula auripennisb L. vibrans Erythemis simplicicollisb

Anaim-AKH Anaim-AKH Anaim-AKH Anaim-AKH Anaim-AKH Anaim-AKH Anaim-AKHa Anaim-AKH Anaim-AKH Anaim-AKHa Libau-AKH Libau-AKH Libau-AKHa Anaim-AKH Anaim-AKHa Anaim-AKHa Libau-AKH Libau-AKH Libau-AKH Libau-AKHa Libau-AKH Libau-AKH Libau-AKHa Libau-AKH Libau-AKHa Libau-AKH Libau-AKH Libau-AKH Libau-AKH Libau-AKH Libau-AKH Libau-AKH Libau-AKHa Erysi-AKH

Gomphidae

Cordulegastridae Macromiidae Corduliidae Libellulidae

a

Assigned solely by retention time in HPLC. Data published previously (see Introduction for references). c Adult and larval specimens were investigated. d Only larval specimens were investigated. b

4.2. AKH sequences and point mutations It may be informative to theorise how ‘‘easy’’ it might be for mutations to arise on the RNA level by

comparing the AKH peptide sequences present in Odonata. Clearly, amino acid substitutions have occurred at positions 2, 5 and 7 from the N terminus in the different peptides (see Table 1). At position 2,

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either a Val or Leu residue is found, while either Ser or Thr is present at position 5, and either Ser or Gly occurs at position 7 (Table 1). All of these changes are simple and conservative point mutations which can be easily achieved and which lead to single base changes when considering the standard genetic code. With this pattern in mind, it is clear that a change from Psein-AKH (with V2T5G7) to Anaim-AKH (V2S5S7) requires two point mutations, whereas only one step is required for the modification of Anaim-AKH to Libau-AKH, and from Libau-AKH to Erysi-AKH. This then, may tentatively be taken as a hint that Anisozygoptera and Anisoptera are more closely related to each other than to Zygoptera. Thus, such data may support the theory that Anisozygoptera and Anisoptera are sister taxa. 4.3. AKH and odonate phylogenies The lack of diversity of AKH neuropeptides in Odonata presents us with only a few character states which, by themselves, would not be sufficient for a phylogenetic analysis. We can, however, use the structural data from the AKH sequences in conjunction with published ideas on phylogenies of Odonata to possibly achieve a high-level phylogeny (see Fig. 2). In the light that Ephemeroptera, which can be used as an outgroup, contain the AKH peptide Anaim-AKH in the only species investigated to date (G. Ga¨de, H.G. Marco and P. Simek, unpublished result), the presence of PseinAKH in all investigated Zygoptera may support the

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phylogenies that this Odonata suborder is separated from the other two odonate suborders. The analysis of more species of Ephemeroptera with respect to the primary sequences of their AKHs is anxiously awaited to strengthen this argument. Our data, however, do not support a paraphyletic state of the Zygoptera as was recently again deduced from mitochondrial 12S ribosomal RNA sequences (Saux et al., 2003). For the remaining two suborders, Anisozygoptera and Anisoptera, we propose Anaim-AKH as the ancestral peptide (see Fig. 2) and support the theory proposed by many authors that these two suborders are sister taxa (Bechly, 1996; Rehn, 2003; Misof and Fleck, 2003). There are a number of phylogenies that are at variance when considering the various families of Anisoptera (see, for example, Fig. 3 in Misof, 2000). Despite a number of variabilities, most authors have Gomphidae plus Aeshnidae and Corduliidae plus Libellulidae closely related (see Misof, 2000). The present work is interpreted in the following way: the ancestral Anaim-AKH is independently and convergently modified to Libau-AKH in the Gomphidae, Corduliidae (i.e. Corduliidae sensu stricto) and Libellulidae; all other investigated families, including the Macromiidae, which some authors keep with the Corduliidae, have the ancestral Anaim-AKH. This is also true for the species Syncordulia gracilis. Its phylogenetic position is not yet clear. Bechly (1996), for example, places the genus Syncordulia into the Austrocorduliidae, which are closely related to the Macromiidae and not to the Corduliidae s.s. Such a

Psein-AKH

ZYGOPTERA Anaim-AKH

ANISOZYGOPTERA

Anaim-AKH

Aeshnidae

?

Petaluridae

Libau-AKH

Gomphidae

Anaim-AKH

Cordulegastridae

Anaim-AKH

Phyllomacromia

Anaim-AKH

Syncordulia

Libau-AKH

Somatochlora

Libau-AKH

Libellulidae

Anaim-AKH

Erysi-AKH

Erythemis

Fig. 2. Phylogeny of Odonata (modified from Misof, 2000) and evolution of their AKH peptides. Note: (a) the investigated species rather than the putative families are given in those cases where phylogenetic relationships are not clear in the literature (Macromiidae, Syncordulidae, etc.) and where one species of the Libellulidae, viz. E. simplicicollis, shows a point mutation; (b) species of the family Petularidae were not investigated in the present study, hence their AKH sequence/identity is still unknown and is depicted by a question mark.

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relationship is also reflected from our peptide data: Phyllomacromia bifasciata and Syncordulia gracilis contain Anaim-AKH and are thus closer related to Cordulegastridae and Aeshnidae, whereas Somatochlora metallica, a Corduliidae s.s., contains Libau-AKH as in the Libellulidae. In one species of Libellulidae, viz. Erythemis simplicicollis, a further conservative modification (change from Val2 to Leu2) has taken place. It is anticipated that more than only this one libellulid species may have this modification, but to date, after analysis of 17 species this is not the case. In conclusion, the sequences of AKH neuropeptides in Odonata cannot be used on their own to distinguish between various phylogenies or to propose an own phylogeny of Odonata. The sequences do, however, offer information to support certain trends in odonate phylogenetic thinking, namely that Anisozygoptera and Anisoptera are sister groups and that, apparently, Corduliidae s.s. and the Libellulidae belong to the most developed phylogenetic taxa. 4.4. Functional role of AKH in Odonata Although four different primary sequences have been found in Odonata, the functional role of AKH has apparently not changed in the different taxa. Each of the four peptides is active, albeit to a different potency, in mobilising lipids in the heterologous bioassay using migratory locusts as the recipient (see Ga¨de et al., 2000) or increasing the concentration of carbohydrates in the haemolymph in a heterologous bioassay using the American cockroach as the recipient (see, for example, Ga¨de, 1990; Ga¨de and Kellner, 1999). Moreover, each of these peptides has been tested in the species in which it was first identified: there it has a pronounced adipokinetic effect (see Introduction); however, in Libellula auripennis a small hypertrehalosaemic effect was also measured (Ga¨de, 1990). In a number of cases, flight experiments have been conducted with dragonfly species, and in each case it was shown that lipid metabolism is paramount (Janssens and Ga¨de, 1993; Rhode, 1998). Taking all these data together, it appears safe to conclude that the peptide’s main metabolic role in, at least the majority (if not all), Odonata is to control lipid metabolism.

Acknowledgements Besides the individuals mentioned in Materials and methods in respect of collection and identification of Odonata, the authors are grateful to a number of other people and organisations who played an essential part during the 13 years in total that it took to bring this project together: Mr. M.P. Janssens (a former MSc student of UCT for collecting a number of South

African odonates, dissecting of glands and help with HPLC), Dr. R. Kellner (Merck AG, Darmstadt, Germany for contributing Edman sequence data for some species), the National Parks Board of the different provinces of South Africa for permission to collect Odonata in various National Parks; Mr. G. Strydom (Kruger National Park) and Mr. Le Riche (Kalahari Gemsbok Park) for logistical support; the Ministry of Agriculture of Mauritius for support during collecting in Mauritius, the Foundation for Research Development which later became the National Research Foundation (Pretoria, South Africa; grant number: 2053806) and the University of Cape Town for financial support.

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